A four-season antiviral mixture multi-component high-efficiency detection method based on capillary electrophoresis

Abstract

The application relates to a kind of four seasons antiviral mixture multi-component high-efficiency detection methods based on capillary electrophoresis, comprising: S1, standard solution preparation;S2, borax buffer preparation;S3, standard curve establishment;S4, sample pretreatment;S5, sample injection detection.The application realizes the high-efficiency separation of four seasons antiviral mixture by high-performance capillary electrophoresis (HPCE), and the sample consumption is less, environment-friendly, suitable for the rapid analysis of multi-component in traditional Chinese medicine complex system.

Description

Technical Field

[0001] This invention belongs to the field of quality detection technology of chemical components of traditional Chinese medicine, and in particular relates to a high-efficiency detection method for multi-components of seasonal antiviral compound based on capillary electrophoresis. Background Technology

[0002] The Four Seasons Antiviral Compound (National Drug Approval Number Z20027669) is composed of 11 traditional Chinese medicines: Houttuynia cordata, Platycodon grandiflorus, Morus alba leaf, Forsythia suspensa, Schizonepeta tenuifolia, Mentha haplocalyx, Perilla frutescens leaf, Prunus armeniaca seed, Phragmites communis rhizome, Chrysanthemum morifolium, and Glycyrrhiza uralensis. It has the functions of clearing heat and detoxifying, reducing inflammation and fever, and is used to treat viral infectious diseases such as upper respiratory tract infections, viral colds, influenza, and mumps.

[0003] Currently, the detection of components such as quercetin, chlorogenic acid, hesperidin, and forsythoside in this preparation is mostly carried out using high-performance liquid chromatography (HPLC) or liquid chromatography-mass spectrometry (LC-MS). Although these methods have high sensitivity, they also have problems such as long analysis time, large consumption of organic solvents, high instrument maintenance costs, and low separation and detection efficiency, which are not conducive to rapid and large-scale application in routine quality control. At the same time, chromatographic analysis methods that rely on a large amount of organic mobile phase will generate a lot of chemical waste, which is inconsistent with the concept of green and sustainable development. Some literature reports that HPLC can simultaneously determine the content of 17 components in the Four Seasons Antiviral Compound, including 2. chlorogenic acid, 10. hesperidin, 13. forsythoside, and 15. quercetin, with an analysis time of up to 60 minutes. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the prior art and provide a high-efficiency detection method for multiple components of the four-season antiviral compound based on capillary electrophoresis. The method achieves efficient separation of the four-season antiviral compound through high-performance capillary electrophoresis (HPCE). The analysis time required for the content of chlorogenic acid, hesperidin, forsythoside and quercetin is only 18 minutes, which is greatly shortened. Moreover, it requires less sample and is environmentally friendly, and is suitable for rapid analysis of multiple components in complex traditional Chinese medicine systems.

[0005] The technical problem solved by this invention is achieved through the following technical solution: A high-efficiency detection method for multi-component antiviral compound preparations based on capillary electrophoresis, the method comprising the following steps: S1. Preparation of standard solutions Weigh 3.54 mg of chlorogenic acid into a 1 ml volumetric flask, dilute to the mark with methanol and water in a 1:1 ratio and shake well to prepare a stock solution with a concentration of 10 mM; take the 10 mM stock solution and dilute it stepwise to the target concentrations of 25, 50, 100, 150 and 200 μM. Weigh 3.02 mg of quercetin into a 2 ml volumetric flask, dilute to the mark with methanol and water in a 1:1 ratio and shake well to obtain a stock solution with a concentration of 5 mM; take the 5 mM stock solution and dilute it stepwise to the target concentrations of 50, 100, 150, 200 and 300 μM. Weigh 5.34 mg of forsythoside into a 2 ml volumetric flask, dilute to the mark with methanol:water 1:1 (10% DMSO for dissolution) and shake well to obtain a stock solution with a concentration of 5 mM; take the 5 mM stock solution and dilute it stepwise to the target concentrations of 50, 100, 150, 200 and 300 μM. Weigh 6.10 mg of hesperidin into a 5 ml volumetric flask, dilute to the mark with methanol:water 1:1 (20% DMSO for dissolution) and shake well to obtain a stock solution with a concentration of 2 mM; take the 2 mM stock solution and dilute it stepwise to the target concentrations of 25, 50, 100, 150 and 200 μM. S2, borax buffer solution preparation Weigh 7.627 g of sodium tetraborate decahydrate, add it to 900 ml of deionized water containing 10% acetonitrile, and sonicate until completely dissolved. Adjust the pH to 9-9.5 with 0.1M HCl. Transfer the pH-adjusted borax solution to a 1000 ml volumetric flask and dilute to the mark to obtain borax buffer solution with a concentration of 20 mM. S3. Establishing the Standard Curve Take 500 μl of each standard solution of chlorogenic acid, quercetin, forsythoside and hesperidin, place them in a sample bottle, and perform CE analysis by capillary electrophoresis to obtain the separation spectra. Linear regression analysis was performed using the standard concentration X and peak area Y. The linear regression equations for chlorogenic acid, quercetin, forsythoside, and hesperidin were y = 0.0934x - 0.3243 (r = 0.9998), y = 0.0735x - 0.4276 (r = 0.9997), y = 0.0545x - 0.697 (r = 0.9997), and y = 0.0364x - 0.0284 (r = 0.9998), respectively. Chlorogenic acid, quercetin, forsythoside, and hesperidin showed good linear relationships with peak area in the concentration ranges of 25–200 μM, 50–300 μM, 50–300 μM, and 25–200 μM, respectively. S4. Sample Pretreatment Take 5.0 ml of different batches of Four Seasons Antiviral Mixture into a 50 ml volumetric flask, dilute to volume with ultrapure water, shake well, filter through a 0.22 μm microporous membrane, and then inject the sample. S5, Precision Test Sample S4 was injected six times consecutively via capillary electrophoresis, yielding RSDs of chlorogenic acid, quercetin, forsythoside, and hesperidin of 2.71%, 1.50%, 1.59%, and 4.62%, respectively. S6, Stability Test One test solution was prepared according to method S4, and capillary electrophoresis was performed at 0, 2, 4, 6, 8, and 12 h. The RSD values ​​of the peak areas of chlorogenic acid, quercetin, forsythoside, and hesperidin were 1.69%, 1.83%, 1.61%, and 3.26%, respectively. S7, Repeatability Test Five samples of the same batch of Four Seasons Antiviral Compound were taken, and test solutions were prepared. The samples were analyzed by capillary electrophoresis. The average contents of chlorogenic acid, quercetin, forsythoside and hesperidin in the antiviral compound were calculated according to the linear equation of S3. The results were 71.66, 229.75, 105.89 and 120.06 μg / ml, respectively, with RSD values ​​of 1.89%, 0.90%, 1.80% and 3.99%, respectively.

[0006] Furthermore, the quartz capillary used in the capillary electrophoresis has an inner diameter of 50 μm and a total length of 60 cm; the capillary electrophoresis uses a 20 mM borax buffer solution with a pH of 9-9.5; the separation voltage of the capillary electrophoresis is +20 kV, the column temperature is 25℃, and the detection wavelength is 254 nm; the pressure injection is 50 mbar × 5 s; before each injection, the capillary is rinsed sequentially with 0.1 M NaOH, water, and buffer solution for 120 s, 120 s, and 180 s; before the first use of the capillary, it is rinsed sequentially with methanol, water, 0.1 M NaOH, water, and buffer solution for 10 min, 5 min, 30 min, 20 min, and 15 min.

[0007] The positive effects that this invention can produce are: 1. High comprehensiveness: It provides a comprehensive new testing standard for some hospital-prepared formulations containing such ingredients, breaking through the limitation of only testing a single ingredient. It establishes an analytical method that can simultaneously determine four key active ingredients: quercetin, chlorogenic acid, hesperidin and forsythoside, realizing the synchronous monitoring of the quality of multiple medicinal materials in the prescription, and significantly improving the comprehensiveness and reliability of quality control.

[0008] 2. High efficiency and speed of analysis: Utilizing the high separation efficiency of high-performance capillary electrophoresis (HPCE), this method can complete the baseline separation and quantification of four components in a short time. The analysis speed is better than that of conventional high-performance liquid chromatography (HPLC), making it more suitable for rapid screening and daily quality control of large batches of samples.

[0009] 3. Environmentally friendly and low operating cost: This method uses an aqueous buffer solution as the main separation medium, consumes very little organic solvent, and generates little chemical waste, which is in line with the concept of green analysis. At the same time, the instrument is relatively simple to maintain, which significantly reduces the reagent and maintenance costs for long-term operation.

[0010] 4. Simple method and small sample volume: This method focuses on the systematic optimization of the buffer system. By comparing different buffer systems, the optimal conditions for capillary zone electrophoresis (CZE) were established. In addition, the sample pretreatment of this method is simple, and no complicated extraction and separation steps are required for direct injection analysis. The required sample volume is extremely small, which improves the analytical efficiency and saves sample costs. Attached Figure Description

[0011] Figure 1 This is the capillary electrophoresis separation spectrum of the forsythoside standard solution of the present invention; Figure 2 This is the capillary electrophoresis separation spectrum of the hesperidin standard solution of the present invention; Figure 3 This is the capillary electrophoretic separation spectrum of the chlorogenic acid standard solution of the present invention; Figure 4 This is a capillary electrophoresis separation spectrum of the quercetin standard solution of the present invention; Figure 5 This is the capillary electrophoresis separation spectrum of the four-season antiviral compound of the present invention; Figure 6 This is a chromatogram for the determination of components in the Four Seasons Antiviral Compound by HPLC. Detailed Implementation

[0012] The present invention will be further described in detail below through specific embodiments. The following embodiments are merely descriptive and not limiting, and should not be used to limit the scope of protection of the present invention.

[0013] An innovative method for the efficient detection of multiple components of a seasonal antiviral compound based on capillary electrophoresis is described, with the following steps: S1. Preparation of standard solutions Weigh 3.54 mg of chlorogenic acid into a 1 ml volumetric flask, dilute to the mark with methanol and water in a 1:1 ratio and shake well to prepare a stock solution with a concentration of 10 mM; take the 10 mM stock solution and dilute it stepwise to the target concentrations of 25, 50, 100, 150 and 200 μM. Weigh 3.02 mg of quercetin into a 2 ml volumetric flask, dilute to the mark with methanol and water in a 1:1 ratio and shake well to obtain a stock solution with a concentration of 5 mM; take the 5 mM stock solution and dilute it stepwise to the target concentrations of 50, 100, 150, 200 and 300 μM. Weigh 5.34 mg of forsythoside into a 2 ml volumetric flask, dilute to the mark with methanol:water 1:1 (10% DMSO for dissolution) and shake well to obtain a stock solution with a concentration of 5 mM; take the 5 mM stock solution and dilute it stepwise to the target concentrations of 50, 100, 150, 200 and 300 μM. Weigh 6.10 mg of hesperidin into a 5 ml volumetric flask, dilute to the mark with methanol:water 1:1 (20% DMSO for dissolution) and shake well to obtain a stock solution with a concentration of 2 mM; take the 2 mM stock solution and serially dilute to the target concentrations of 25, 50, 100, 150 and 200 μM. S2, borax buffer solution preparation Weigh 7.627 g of sodium tetraborate decahydrate, add it to 900 ml of deionized water containing 10% acetonitrile, and sonicate until completely dissolved. Adjust the pH to 9.40 using 0.1 M HCl. Transfer the pH-adjusted borax solution to a 1000 ml volumetric flask and dilute to the mark to obtain borax buffer solution with a concentration of 20 mM. S3. Establishing the Standard Curve Take 500 μl of each standard solution sample from the concentrations of chlorogenic acid, quercetin, forsythoside, and hesperidin, place them in a sample vial, and perform CE analysis by capillary electrophoresis to obtain the respective separation spectra, as shown below. Figures 1-4 As shown; Linear regression analysis was performed using the standard concentration X and peak area Y. The linear regression equations for chlorogenic acid, quercetin, forsythoside, and hesperidin were y = 0.0934x - 0.3243 (r = 0.9998), y = 0.0735x - 0.4276 (r = 0.9997), y = 0.0545x - 0.697 (r = 0.9997), and y = 0.0364x - 0.0284 (r = 0.9998), respectively. Chlorogenic acid, quercetin, forsythoside, and hesperidin showed good linear relationships with peak area in the concentration ranges of 25–200 μM, 50–300 μM, 50–300 μM, and 25–200 μM, respectively. S4. Sample Pretreatment Take 5.0 ml of different batches of Four Seasons Antiviral Mixture into a 50 ml volumetric flask, dilute to volume with ultrapure water, shake well, filter through a 0.22 μm microporous membrane, and then inject the sample. S5, Precision Test Sample S4 was injected six times consecutively via capillary electrophoresis, yielding RSDs of chlorogenic acid, quercetin, forsythoside, and hesperidin of 2.71%, 1.50%, 1.59%, and 4.62%, respectively. S6, Stability Test One test solution was prepared according to method S4, and capillary electrophoresis was performed at 0, 2, 4, 6, 8, and 12 h. The RSD values ​​of the peak areas of chlorogenic acid, quercetin, forsythoside, and hesperidin were 1.69%, 1.83%, 1.61%, and 3.26%, respectively. S7, Repeatability Test Five samples of the same batch of Four Seasons Antiviral Compound were taken, and test solutions were prepared. The samples were analyzed by capillary electrophoresis. The average contents of chlorogenic acid, quercetin, forsythoside and hesperidin in the antiviral compound were calculated according to the linear equation of S3. The results were 71.66, 229.75, 105.89 and 120.06 μg / ml, respectively, with RSD values ​​of 1.89%, 0.90%, 1.80% and 3.99%, respectively.

[0014] The quartz capillary used for capillary electrophoresis has an inner diameter of 50 μm and a total length of 60 cm. The capillary electrophoresis uses a 20 mM borax buffer solution with a pH of 9.40. The separation voltage is +20 kV, the column temperature is 25℃, and the detection wavelength is 254 nm. The injection pressure is 50 mbar × 5 s. Before each injection, the capillary is rinsed sequentially with 0.1 M NaOH, water, and buffer solution for 120 s, 120 s, and 180 s. Before the first use, the capillary is rinsed sequentially with methanol, water, 0.1 M NaOH, water, and buffer solution for 10 min, 5 min, 30 min, 20 min, and 15 min.

[0015] Take 500 μL of the sample treated with S4 of this invention, place it in a sample vial, and perform CE analysis according to the capillary electrophoresis conditions described above. The result is as follows: Figure 5 The separation spectra shown are illustrated. Under the optimized electrophoresis conditions described above, quercetin, chlorogenic acid, hesperidin, and forsythoside were baseline separated within 18 minutes, with a separation time of only 18 minutes. The four components exhibited good linearity within their respective concentration ranges (chlorogenic acid and hesperidin: 25–200 μM; quercetin and forsythoside: 50–300 μM) (r ≥ 0.9976). Six consecutive injections of the same test solution yielded RSDs of 2.71%, 1.50%, 1.59%, and 4.62% for the peak areas of chlorogenic acid, quercetin, forsythoside, and hesperidin, respectively, indicating good method precision. In the repeatability test (n=5), the average contents of the four components in the Four Seasons Antiviral Compound were measured to be 71.66, 229.75, 105.89, and 120.06 μg / ml, respectively, with RSD values ​​of 1.89%, 0.90%, 1.80%, and 3.99%, indicating good method repeatability. The sample solution showed good stability within 12 hours (RSD of peak area for each component ≤ 3.26%).

[0016] The method has been verified to be applicable to the quantitative determination of chlorogenic acid, quercetin, forsythoside and hesperidin in the Four Seasons Antiviral Compound.

[0017] like Figure 6 The contents of 17 components in the Four Seasons Antiviral Compound were simultaneously determined by HPLC: 2. chlorogenic acid, 10. hesperidin, 13. forsythoside, and 15. quercetin. The analysis time was 60 min.

[0018] This invention employs a borax buffer system to achieve efficient separation of four target compounds. Its core mechanism lies in utilizing borate ions (B(OH)4). - Selective complexation between hydroxyl groups with different structures induces differential changes in the charge state of the solute; in capillary electrophoresis, the apparent migration rate of a compound is affected by the vector superposition of electroosmotic flow (EOF) and its own effective electrophoretic flow. All four compounds contain multiple hydroxyl groups, including different structural types such as cis-ortho-dihydroxy, trans-ortho-hydroxy, and phenolic hydroxyl groups. Borate ions (B(OH)4) - It can undergo a reversible complexation reaction with the cis-ortho-dihydroxy group in the molecule to generate a negatively charged borate ester complex, thereby significantly altering the charge state and electrophoretic behavior of the solute.

[0019] Although forsythoside molecule contains ortho-hydroxyl groups, it does not have a cis-ortho-dihydroxyl structure that can complex with borate ions. It is electrically neutral as a whole and migrates only with electroosmotic flow during the separation process. Therefore, it has the fastest migration speed and is the first to emerge as a peak.

[0020] The glucose unit in the hesperidin molecule contains a group of cis-ortho-dihydroxy groups, which can complex with borate ions; at the same time, its aglycone contains free phenolic hydroxyl groups, which can carry a small amount of negative charge in the buffer system of this patent, generating a tendency to migrate towards the anode by electrophoresis, partially offsetting the driving effect of electroosmotic flow. Therefore, the migration speed is slower than that of forsythoside, and the migration time is correspondingly longer.

[0021] Chlorogenic acid contains a carboxyl group, which can be completely ionized and carry a negative charge in the borax buffer system. Its quinic acid fragment contains cis-ortho-dihydroxyl groups, and its caffeic acid fragment contains ortho-dihydroxyl groups, both of which can complex with borate ions, significantly enhancing the overall negative charge. The counteracting effect on electroosmotic flow is significantly greater than that of hesperidin, thus resulting in a longer migration time.

[0022] The ortho-dihydroxyl groups on the B ring of quercetin can form strong complexes with borate ions. The phenolic hydroxyl groups at other positions in the molecule can be completely ionized in the existing system, carrying multiple negative charges, which significantly counteract the electroosmotic flow. Simultaneously, quercetin lacks glycosyl groups, has a small molecular radius, and possesses an extremely high effective charge density. The resulting reverse electrophoretic resistance maximally counteracts the driving force of the electroosmotic flow, ultimately resulting in the slowest migration speed and the latest peak elution time.

[0023] Although embodiments of the invention have been disclosed for illustrative purposes, those skilled in the art will understand that various substitutions, variations, and modifications are possible without departing from the spirit and scope of the invention and the appended claims. Therefore, the scope of the invention is not limited to the contents disclosed in the embodiments.

Claims

1. A high-efficiency detection method for multi-component antiviral compound preparations based on capillary electrophoresis, characterized in that: The steps of the method are as follows: S1. Preparation of standard solutions Weigh 3.54 mg of chlorogenic acid into a 1 ml volumetric flask, dilute to the mark with methanol and water in a 1:1 ratio and shake well to prepare a stock solution with a concentration of 10 mM; take the 10 mM stock solution and dilute it stepwise to the target concentrations of 25, 50, 100, 150 and 200 μM. Weigh 3.02 mg of quercetin into a 2 ml volumetric flask, dilute to the mark with methanol and water in a 1:1 ratio and shake well to obtain a stock solution with a concentration of 5 mM; take the 5 mM stock solution and dilute it stepwise to the target concentrations of 50, 100, 150, 200 and 300 μM. Weigh 5.34 mg of forsythoside into a 2 ml volumetric flask, dilute to the mark with methanol:water 1:1 (10% DMSO for dissolution) and shake well to obtain a stock solution with a concentration of 5 mM; take the 5 mM stock solution and dilute it stepwise to the target concentrations of 50, 100, 150, 200 and 300 μM. Weigh 6.10 mg of hesperidin into a 5 ml volumetric flask, dilute to the mark with methanol:water 1:1 (20% DMSO for dissolution) and shake well to obtain a stock solution with a concentration of 2 mM; take the 2 mM stock solution and dilute it stepwise to the target concentrations of 25, 50, 100, 150 and 200 μM. S2, borax buffer solution preparation Weigh 7.627 g of sodium tetraborate decahydrate, add it to 900 ml of deionized water containing 10% acetonitrile, and sonicate until completely dissolved. Adjust the pH to 9-9.5 with 0.1M HCl. Transfer the pH-adjusted borax solution to a 1000 ml volumetric flask and dilute to the mark to obtain borax buffer solution with a concentration of 20 mM. S3. Establishing the Standard Curve Take 500 μl of each standard solution of chlorogenic acid, quercetin, forsythoside and hesperidin, place them in a sample bottle, and perform CE analysis by capillary electrophoresis to obtain the separation spectra. Linear regression analysis was performed using the standard concentration X and peak area Y. The linear regression equations for chlorogenic acid, quercetin, forsythoside, and hesperidin were y = 0.0934x - 0.3243 (r = 0.9998), y = 0.0735x - 0.4276 (r = 0.9997), y = 0.0545x - 0.697 (r = 0.9997), and y = 0.0364x - 0.0284 (r = 0.9998), respectively. Chlorogenic acid, quercetin, forsythoside, and hesperidin showed good linear relationships with peak area in the concentration ranges of 25–200 μM, 50–300 μM, 50–300 μM, and 25–200 μM, respectively. S4. Sample Pretreatment Take 5.0 ml of different batches of Four Seasons Antiviral Mixture into a 50 ml volumetric flask, dilute to volume with ultrapure water, shake well, filter through a 0.22 μm microporous membrane, and then inject the sample. S5, Precision Test The S4 sample was injected six times consecutively via capillary electrophoresis, and the RSDs of the areas for chlorogenic acid, quercetin, forsythoside, and hesperidin were 2.71%, 1.50%, 1.59%, and 4.62%, respectively. S6, Stability Test One test solution was prepared according to method S4, and capillary electrophoresis was performed at 0, 2, 4, 6, 8, and 12 h. The RSD values ​​of the peak areas of chlorogenic acid, quercetin, forsythoside, and hesperidin were 1.69%, 1.83%, 1.61%, and 3.26%, respectively. S7, Repeatability Test Five samples of the same batch of Four Seasons Antiviral Compound were taken, and test solutions were prepared. The samples were analyzed by capillary electrophoresis. The average contents of chlorogenic acid, quercetin, forsythoside and hesperidin in the antiviral compound were calculated according to the linear equation of S3. The results were 71.66, 229.75, 105.89 and 120.06 μg / ml, respectively, with RSD values ​​of 1.89%, 0.90%, 1.80% and 3.99%, respectively.

2. The method for high-efficiency detection of multi-component antiviral compound based on capillary electrophoresis according to claim 1, characterized in that: The quartz capillary used for capillary electrophoresis has an inner diameter of 50 μm and a total length of 60 cm. The capillary electrophoresis uses a 20 mM borax buffer solution with a pH of 9–9.

5. The separation voltage is +20 kV, the column temperature is 25℃, and the detection wavelength is 254 nm. The injection pressure is 50 mbar × 5 s. Before each injection, the capillary is rinsed sequentially with 0.1 M NaOH, water, and buffer solution for 120 s, 120 s, and 180 s. Before the first use, the capillary is rinsed sequentially with methanol, water, 0.1 M NaOH, water, and buffer solution for 10 min, 5 min, 30 min, 20 min, and 15 min.

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